Dried Blood Spot Postmortem Metabolic Autopsy With Genotype Validation for Sudden Unexpected Deaths in Infancy and Childhood in Hong Kong

Background Inborn errors of metabolism (IEM) are collectively rare but potentially preventable causes of sudden unexpected death (SUD) in infancy or childhood, and metabolic autopsy serves as the final tool for establishing the diagnosis. We conducted a retrospective review of the metabolic and molecular autopsy on SUD and characterized the biochemical and genetic findings. Methodology A retrospective review of postmortem metabolic investigations (dried blood spot acylcarnitines and amino acid analysis, urine metabolic profiling where available, and next-generation sequencing on a panel of 75 IEM genes) performed for infants and children who presented with SUD between October 2016 and December 2021 with inconclusive autopsy findings or autopsy features suspicious of underlying IEM in our locality was conducted. Clinical and autopsy findings were reviewed for each case. Results A total of 43 infants and children aged between zero days to 10 years at the time of death were referred to the authors’ laboratories throughout the study period. One positive case of multiple acyl-CoA dehydrogenase deficiency was diagnosed. Postmortem reference intervals for dried blood spot amino acids and acylcarnitines profile were established based on the results from the remaining patients. Conclusions Our study confirmed the importance of metabolic autopsy and the advantages of incorporating biochemical and genetic testing in this setting.


Introduction
Many inborn errors of metabolism (IEM) are known to present with sudden unexpected death (SUD) in infancy and childhood.Well-known examples include fatty acid oxidation defects, urea cycle disorders, organic acidurias, and disorders of galactose and fructose metabolism [1].It was estimated from previous studies that metabolic disorders account for 3-5% of SUD cases [2,3].Often these patients remain asymptomatic and well before the acute fatal decompensation of their metabolic disorder.As many of these conditions are treatable if diagnosed early, expanded newborn screening for IEM has been advocated worldwide as a cost-effective screening approach and has been implemented in our locality stepwise since 2015.In 2020, a government-funded newborn screening program for a panel of 26 IEMs, including disorders of amino acid, disorders of organic acids, disorders of fatty acid oxidation, and other IEMs, was implemented in all public hospitals with maternity services [4].The service, however, does not cover babies born in the private sector.Nevertheless, metabolic investigations have long been recommended as part of routine postmortem examination for SUDs in infancy and childhood by various guidelines and protocols [1,5].Recommended tests include acylcarnitine and amino acid analysis by tandem mass spectrometry on dried blood spot and bile specimens [1,[6][7][8][9][10], organic acid analysis in urine or vitreous specimens [11], as well as enzymatic and other functional studies on tissue specimens such as skin, muscle, and liver biopsy [3,10,12].Recently, the use of massively parallel sequencing in postmortem DNA samples has also been reported as an alternative diagnostic approach [13,14].
The previous local report on the causes of unexpected deaths in children under two years of age was published in 2006 [15].IEM was identified as the cause of death in five cases (2.7%) out of 183 cases of sudden deaths recorded throughout the four-year study period between 1997 and 2002.In 2010, our group also encountered a previously well 14-year-old Chinese boy who presented with SUD in his adolescence [16].Postmortem investigations by biochemical and molecular testing confirmed the diagnosis of multiple acyl- CoA dehydrogenase deficiency, or glutaric aciduria type II.Cascade family screening enabled accurate risk assessment in other family members and avoided unnecessary treatments in unaffected members.The importance of postmortem metabolic investigations was again exemplified by this case.Thus, in 2016, our group began providing metabolic investigation to infants and children presenting with SUD.Postmortem samples for metabolic investigations were collected at autopsy in selected cases with inconclusive autopsy findings or patients with autopsy findings suggestive of a metabolic disorder, such as fatty infiltration of the liver.Tests provided included dried blood spots for acylcarnitines and amino acid analysis, urine organic acids, metabolic profiling, and next-generation sequencing with an IEM gene panel.This study aims to retrospectively review the results of all postmortem metabolic investigations performed at our laboratories to improve the understanding of the local epidemiology and establish reference intervals for the interpretation of acylcarnitines and amino acids on postmortem dried blood spots.

Methods
Postmortem dried blood spot samples collected on Whatman 903 filter paper were subjected to amino acid and acylcarnitines analyses using the Neobase non-derivatized MSMS Kit (Perkin Elmer, Waltham, MA, US) on the Waters Acquity TQD LC-MS/MS system (Waters, Milford, MA, US).A dried blood disk (3.2 mm in diameter) was punched out for extraction and the eluate was injected into liquid chromatography-mass spectrometry (LC-MS)/mass spectrometry (MS) directly without derivatization and chromatography.The target amino acids and acylcarnitines were detected and quantified using the multiple reaction monitoring mode.Urine multi-target metabolic profiling was performed with an in-house dilute-and-shoot LC-MS/MS protocol using gradient high-pressure liquid chromatography (Agilent Zorbax Eclipse AAA 4.6 mm × 15 cm, 5 µm reversed-phase column) and electrospray ionization on the Waters Xevo TQ-MS System (Waters, Milford, MA, US).The method allows simultaneous analyses of selected organic acids, acylglycines, acylcarnitines, amino acids, purines, pyrimidines, and other relevant markers in IEM by multiple reaction monitoring analysis.Next-generation sequencing was performed on the genomic DNA extracted from the dried blood spot specimen using a custom amplicon-based IEM panel on the iSeq Sequencing System (Illumina, Inc, San Diego, CA, US), details of which had been previously published [17].Data analysis was limited to 75 genes (Table 1) which are causative of conditions that may present with SUD.Pathogenicity of variants was classified according to the standards and guidelines for the interpretation of sequence variants published by the American College of Medical Genetics and Genomics in 2015 [18].

Statistical analysis
Postmortem reference intervals for dried blood spot amino acids and acylcarnitine profile results were established based on profiles from patients without IEM identifiable in the aforementioned IEM gene panel.Outliers were excluded by the Reed method [19], and the non-parametric method was used to derive the central 95% reference intervals [20].Statistical analysis was performed using MedCalc® Statistical Software version 20.106 (MedCalc Software Ltd, Ostend, Belgium; https://www.medcalc.org;2022).

Patient demographics and autopsy findings
Throughout the study period from 2016 to 2021, a total of 43 infants and children who presented with SUD were referred to the authors' laboratories, including 19 female and 24 male patients (Table 2 and Table in the Appendices).The majority of patients (72%) were aged between 30 days to 12 months, three (7%) were under one month, and nine (21%) were above one year.Among these patients, 18 (42%) had previously undergone newborn screening for inborn errors of metabolism.The cause of death was ascertained in 12 patients.Details of each case are presented in

Metabolic investigation results and positive case
Dried blood spot specimens were received for all patients referred to our laboratories.Urine specimens were available in only six cases, heparin plasma in 24 cases, and muscle biopsy in six cases.The specimens, including blood and urine samples, were collected at the time of autopsy, which ranged from one to three days postmortem.The specimens were stored at -20°C with a desiccator until the time of analysis.
Out of all cases referred, acute decompensation of a metabolic disorder was identified as the cause of death in one female patient (patient 11 in Table 4 in the Appendices).The patient was born full-term to nonconsanguineous Chinese parents with an unremarkable perinatal history and past medical history.There was no known family history of metabolic diseases.She had not undergone expanded newborn screening for IEM at birth.She presented at 16 months of age with fever and upper respiratory tract symptoms.She was noted to have on-and-off twitching with loss of consciousness at home before being admitted to the emergency department.On arrival at the hospital, she developed cardiac arrest.The blood glucose meter onspot showed hypoglycemia.Unfortunately, the patient succumbed despite resuscitation and intravenous glucose infusion.Parainfluenzae group 3 direct immunofluorescence and parainfluenza virus 3 RNA was later confirmed positive on nasopharyngeal swabs.Urine metabolic profiling detected significant dicarboxylic aciduria, including glutaric acid, 2-hydroxyglutaric acid, adipic acid, ethylmalonic acid, and methylsuccinic acid, along with marked hyperexcretion of suberylglycine, hexanoylglycine, isovalerylglycine, isobutyrylglycine.Dried blood spot metabolic autopsy was remarkable for a very low level of free carnitine than expected for postmortem specimens.The plasma acylcarnitine profile showed generalized elevations of C4 to C18 acylcarnitines.Next-generation sequencing with the aforementioned IEM gene panel revealed two heterozygous missense variants in the electron transfer flavoprotein dehydrogenase (ETFDH) gene, i.e., NM_004453.4:c.1601C>Tp.(Pro534Leu) and NM_004453.4:c.1669G>Ap. (Glu557Lys), which were also identified by Sanger sequencing with compound heterozygosity of the two variants confirmed by parental genotyping.The first missense variant c.1601C>T is predicted to cause the substitution of proline at residue 534 with leucine at the docking site of electron transport flavoprotein within the electron transfer flavoprotein-ubiquinone oxidoreductase domain [21].The variant has been reported in multiple patients with multiple acyl-CoA dehydrogenase deficiency [21][22][23][24].Significantly reduced ETFDH protein and deficiencies of oxidative phosphorylation complexes II and III in liver homogenate were demonstrated in compound heterozygous patients harboring the variant [21,22].The variant has an allele frequency of 0.0024% (6/251308) globally but is absent in controls among East Asians according to the Genome Aggregation Database (gnomAD v2.1.1).It is listed as a disease-causing mutation in Human Gene Mutation Database Professional 2022.1 (CM081237) and a pathogenic variant in ClinVar (RCV000483304.1).The second missense variant c.1669G>A is predicted to cause substitution of glutamic acid with lysine at residue 557 within the electron transfer flavoprotein-ubiquinone oxidoreductase domain, with an allele frequency of 0.0008% (2/250922) globally and 0.0054% (1/18392) among East Asians according to the Genome Aggregation Database (gnomAD v2.1.1).The variant is predicted to be damaging (SIFT/PROVEAN/MetaSVM) and probably damaging (PolyPhen-2) according to in silico analyses.It has been listed as having uncertain significance in ClinVar (VCV000971109.2).Overall, the two variants were considered pathogenic and likely pathogenic, respectively.A diagnosis of multiple acyl-CoA dehydrogenase deficiencies was made based on the biochemical and molecular findings and cascade family screening was arranged.
In the remaining 42 patients, dried blood spot metabolic autopsy and/or urine metabolic profiling did not reveal pathological patterns.Next-generation sequencing with the IEM gene panel in the remaining patients identified three heterozygous likely pathogenic or pathogenic variants in two patients (details in Table 4 in the Appendices).The three variants include (1) NM_001085411.3) mitochondrial DNA depletion syndrome 5 (encephalomyopathy with or without methylmalonic aciduria), respectively, and all three phenotypes are autosomal recessively inherited.As there were no corresponding clinical and biochemical features identified in these two patients and no other pathogenic variants were detected by the IEM gene panel, the two cases were considered non-IEM.The range of results and reference intervals of acylcarnitine and amino acid profile in postmortem dried blood spot samples in the remaining patients were presented in Table 3.

Discussion
This is the first local report on metabolic autopsy combining biochemical and genetic approaches.We have reported the range of acylcarnitine and amino acid levels in postmortem dried blood spots from a group of patients who had been genetically confirmed to be unaffected by any of the known IEMs covered by our gene panel.Our findings were similar to the previous reports, which showed a gross increase in all amino acids and acylcarnitines in dried blood spots obtained in the postmortem period [8,9].Based on the experience of these authors, diagnosis for metabolic disease in the postmortem period based on dried blood spot metabolic profile is still possible by recognizing a marked increase in a particular disease marker relative to the background increase in other amino acid or acylcarnitine species.We, however, encountered practical difficulties with this approach as the number of fold changes in certain analytes relative to the others to be regarded as significant is poorly defined.Furthermore, some IEM may not have a specific diagnostic marker on dried blood spot metabolic profile.For instance, the postmortem dried blood spot profile of the positive case identified in our study showed a markedly low level of free carnitine, but the characteristic elevation of multiple acylcarnitine species was not evident compared with other postmortem samples, as it overlapped with the typical postmortem generalized increases in multiple acylcarnitine species.Even the use of derived indices combining multiple markers used in living patients, such as the glutaric aciduria-II index [25] did not improve the diagnostic performance.This problem was also encountered in the previous fatal multiple acyl-CoA dehydrogenase deficiency case diagnosed by our group [16].
Organic acid analysis is another important pillar in the postmortem diagnosis of IEM.Both urine and vitreous humor have been reported as suitable for organic acid analysis in autopsy settings [1,11].In our positive case, metabolite characteristics of multiple acyl-CoA dehydrogenase deficiency were identified in the urine specimen.Although urine organic acids and metabolic profiling are powerful investigations, urine specimens are often not available or only obtained in scant volumes at the time of autopsy as exemplified in our cohort of patients.Vitreous humor was also not available for metabolic investigations in our local protocol as the volume is limited in children and especially infants and is often reserved for other important investigations such as toxicological testing.
Genetic and genomic testing is an important adjunct to the above diagnostic modalities from our experience.DNA can be extracted from specimens that are readily available at autopsy and are stable in the postmortem period.Uncertain biochemical findings can be verified with the sequencing findings, while biochemical data can aid in variant interpretation, especially when variants of uncertain significance are encountered.Simultaneous analysis of multiple IEM genes by next-generation sequencing enhances the cost-effectiveness and practicality of this approach, while the use of a targeted gene panel, as opposed to exome sequencing, reduces the number of variants requiring interpretation and further lowers the cost of analysis.
Throughout the five-year study period, our group had only identified one positive case, fewer than that reported in the previous local study which reported five IEM cases throughout a four-year period between 1999 and 2002 [15].The apparent reduction in the number of IEM cases diagnosed in this setting may be related to the territory-wide provision of expanded newborn screening of IEM introduced in 2015 [4].Despite the continuous decline in IEM cases among infants and children presenting with SUD is expected, the metabolic investigation is still an essential part of the autopsy, as patients with late-onset or atypical forms of IEM may not be detected at newborn screening.
Finally, there is still a significant proportion of patients in our cohort with cause of death unidentified after thorough postmortem examinations.Our current analysis focused only on metabolic causes of death.Other mendelian diseases that may present with SUD with negative autopsy findings have not been excluded, including cardiac diseases, such as channelopathies and cardiomyopathies, which have been reported to account for 16% to 34% of SUDs in infants and children [26,27].Future directions to delineate the cause of death in this group of patients would include panel testing for these cardiac conditions, which may hopefully prevent mortalities in affected families.

Conclusions
We have reported our five-year experience in the metabolic and genetic autopsy for IEM of children and infants who presented with SUD and have defined the reference intervals for plasma acylcarnitines and amino acids in postmortem dried blood spots based on reference subjects genetically confirmed to be unaffected.We have presented one positive case identified by our group, a case of multiple acyl-CoA dehydrogenase deficiency, which was diagnosed based on the urine metabolic profile and genetic testing.
Although postmortem dried blood acylcarnitine and amino acid profile had long been used to screen for fatty acid oxidation disorders and other IEM, our case highlights the diagnostic difficulty of interpreting postmortem dried blood spots, especially in the case of multiple acyl-CoA dehydrogenase deficiency.Our study confirmed the importance of metabolic autopsy and the advantages of incorporating both biochemical and genetic testing in this setting.

NBSIEM = newborn screening
for inborn errors of metabolism; IEM = inborn errors of metabolism; FT = full term; NSD = normal spontaneous delivery; CS = cesarean section; AN = antenatal; PN = postnatal; NICU = neonatal intensive care unit *: Age range: Preterm neonatal = the period at birth when a newborn is born before the full gestation period Term neonatal birth = 27 days Infancy 28 days = 12 months Toddler 13 months = 2 years Early childhood = 2-5 years Middle childhood = 6-11 years All cases of infants and children under the age of 18 who presented with SUD in Hong Kong between October 2016 and December 2021 referred to the authors' laboratories for metabolic investigations were retrospectively reviewed.Clinical notes and perimortem laboratory investigations on initial presentation where available were retrieved from hospital electronic patient records.Autopsy records and results of postmortem laboratory investigations, including metabolic analyses, were retrieved from laboratory databases.This study was approved by the Hospital Authority Kowloon West Cluster Research Ethics Committee (KW/EX-21-060(158-03)), Hong Kong Children's Hospital Research Ethics Committee (HKCH-REC-2021-052), and Ethics Committee of the Department of Health (L/M 110/2021) (complying with local research ethical review regulation based on seven hospital clusters and the Department of Health).

Table 4
in the Appendices.